Oxidation of sterols



Patented Apia. 25, i950 N lTED STATES PATENT OFFICE to E. I. duPont dc Nemours & Company, Wilmington, Del., a corporation of Delaware No'Drawing. Application October 14, 1947, Serial No. 779,835

16 Claims.

This invention relates to improvements in the process for effectingoxidation of 3-hydroxy-A sterols to produce the'koxo derivatives. The invention is particularl directed to the oxidation of cholesterol and related sterols which are valuable intermediates in the synthesis of Vitamin D.

Variousmethods have been described for effecting the selective oxidation of cholesterol and other sterols when in the form of their esters or similar derivatives which can be converted to an hydroxyl group and whichprotect it from chemical attack during the conversion of the sterol to the 7-oxo derivative.

Heretofore, the sterol derivative, such as an ester including the chloride, isdissolvedin acetic acid and subjected to the oxidizing influence of chromic acid anhydride (CrOa) which is customarily dissolved in a small amount of Water and diluted with additional acetic acid since the chromic acid anhydride itself is very soluble in water and almost insoluble in acetic acid. In this manner, a reaction medium consisting of chromic acid anhydride dissolved in high percentage aqueous acetic acid of'from 90% to"98% is obtained. In carrying out these oxidations, however, certain side reactions occur along with the oxidation of the sterol to the 7-oxo derivative, resulting in relatively small yields of the desired product and the use of an excessive amount of the chromic acid anhydride. Several processes have been described for carrying out these oxidations, such as found in -Windaus, Ann. 520, 192 (1935), see also U. S. P. 2,098,984; U. S. P. 2,215,727 to Rosenberg & Tinker; U. S. P. 2,237,762 to Marker and British Patent 558,361 to Boots. In the Windaus, Rosenberg and Marker processes the oxidation is carried out with chromic acid anhydride in aqueousacetic acid, as described above, and the yields of the resulting '7-oxo derivative are, in each case, approximately 25% of theory. In the process of the Boots patent it was found that the yield could be materially increasedif, in placeof the aqueous acetic acid formerly used, acetic acidanhydride were substituted and the oxidation carried out with chromic acid anhydride for which acetic acid anhydride is a solvent. This process reduces the number of side reactions and effects a somewhat'betterutilization of the chromic acid anhydride. Inthis process a yield of from 34% to 38% of 7-oxo chloesterol acetate was obtained.

These processes are dangerous to operate; espe* cially where a solution of thechromic acid anhydride in acetic acid anhydride is employed, for such mixture ignites spontaneously when warmed to 90 C. andthe aqueous acetic 'acidsolution of the chromic acid anhydride decomposes violently at C.-so thatextreme care must be employed in carrying out theprocesses heretofore described for effecting oxidation of the sterol compounds to the'7-oxoderivatives. Theyare wasteful of materials because-the chemical attack is notspecifically directed to -the T-position of thesterol molecule, causing loss of sterol as well as oxidizing agent. In the process described by Windaus, for instance; only 11 of the chromic acid anhydride is used toform -the desired 7-oxo derivative and in the process of- British'Patent 558,361 only 23% is thus utilized, whilethe-remainder is consumed in side reactions.

It is an object of thisinvention to provide a process for the'oxidation of sterolsto-their 7-oxo derivatives which gives materially increased yields by means of providing a novel oxidation medium for said sterols which restricts oxidation almost. entirely tothe desired .7-position with resulting economy of sterol. and oxidizing agent. It provides a process that is simple and economical to operate, andwhich is less hazardous than the processes previously employed.

I have found that 3-hydroxy-A -sterol derivatives, of which class cholesteryl acetate is employed as a typical example, can be oxidized by means of metalsalts of hexavalent chromium oxide such as the metal chromates including d1- chromates, to give improved' 'yields of the7-oxo sterol derivatives. The oxidation can be carried'out in a medium consisting either of aqueous acetic acid or of acetic acid containing acetic'aci'd anhydride. In thisprocessthe highest yields are obtained under anhydrous conditions, that is, where an excess of acetic anhydride is maintained throughoutlthe oxidation. Even better yields of the 7-oxoisterol derivatives canlbe obtained when the oxidation with a metal chromate is carried outin the presenceof air or oxygen, since I have found that, unexpectedly, molecular oxygen under' these conditions is an excellent oxidizing agent "for the'pro'diiction 'of fT-oxo derivatives. Furthermore, by the addition'ofair or oxygen the amount of the metal :chromate required is materially reduced. It is also desirable to carryout the oxidation in the presence of added alkali metalacetate or similar metal sa'lt of an organic carboxylic acid, as it has the effect of further increasing the desired 7-oxo derivatives.

The following examples are givento illustrate the invention. The parts-usedare by weight.

Example 1 42.8 parts (:10 incl) of cholesteryl acetate, melting point 114-115 0., are dissolved in 120 parts of glacial acetic acid and '70 parts of acetic acid anhydride by warming in a stoppered vessel to 30-35 C. 25.9 parts of sodium chromate, Na2CrO4, (.16 mol, equivalent to 120% of the calculated amount) are now dissolved in the mixture under agitation and the temperature is maintained at 30-40 C. for 48 hours, preferably while agitating. The yellow color of the sodium chromate solution gradually gives way to the clear green color of chromic oxide (Cr203) and the progress of the oxidation can be conveniently estimated by analyzing for the remaining hexavalent chromium. When not more than about of hexavalent chromium is left, the reaction mass, which is a green colored magma of 'l-oxo cholesteryl acetate, is diluted with 40 parts of ice to decompose the acetic anhydride and to complete the precipitation of 'l-oxo cholesteryl acetate. The shiny crystals are filtered on a nutch and washed with methanol and water alternately until the crystals are colorless. Yield: 26 parts, melting point 153-155 C. By adding part of the methanol-water washes to the initial filtrate until turbidity sets in, a secondary crop may be obtained with a yield of 3 parts and melting point 145-150 C. The crude 'l-oxo chloesteryl acetate can be readily purified by crystallization from low boiling petroleum fractions, whereby there is obtained 24.3 parts of 'l-oxo cholesteryl acetate, melting point 160-161 C. (55% of theory) and 1.3 parts of unchanged cholesteryl acetate (3% of theory); yield based on cholesteryl acetate consumed: 58% of theory.

Example 2 If, in Example 1, 40 parts of benzene or trichlorethylene or similar solvent stable to oxidation by chromates are added to the oxidation mass, the reaction time is shortened because the added solvent helps to keep the reaction mass homogeneous. If desired, the 7-oxo choleteryl acetate may be isolated by adding a further amount of the above solvent at the end of the oxidation, and extracting the 'l-oxo cholesteryl acetate by means of the solvent from the dilute aqueous-acetic acid medium. After washing of the solvent layer with water, the 'l-oxo cholesteryl acetate is obtained by adding methanol and filtering of the well-formed crystals. The yield obtained is similar to that of Example 1, and the crude '7-oxo cholesteryl acetate has a melting point of '7-160 C.

Example 3 The 42.8 parts of cholesteryl acetate in Example 1 or 2 may be replaced by 38.6 parts (.10 mol) of cholesterol, if the cholesterol is first refiuxed at 120-125 C. for one hour with 70 parts of acetic acid anhydride. cholesteryl acetate is thus produced in situ and substantially identical yields of 7-oxo cholesteryl acetate are obtained when this solution is oxidized as in either Example 1 or Example 2.

Example 4 If desired, a sodium chromate solution can be prepared from chromic acid anhydride and soda ash in situ in the following manner;

1'7 parts of soda ash .16 mol) are added gradually to a mixture of 80 parts of glacial acetic acid and 50 parts of acetic acid anhydride. At a temperature of from 50 to 70 C. the soda ash dissolves rapidly under evolution of CO2, and with formation of sodium acetate dissolved in acetic acid. 16 parts of chromic acid anhydride,

CrOa (.16 mol), are added to the solution. The red color of ClOs changes at once to the bright yellow color of NazCrO4. After cooling the solution to 30-35 0., 42.8 parts of cholesteryl acetate (.10 mol) are added, dissolved in 40 parts of acetic acid and 40 parts of acetic acid anhydride, and the oxidation is carried out as in Example 1.

Example 5 If, in Example 4, the amount of soda ash is increased, the reaction mass will contain sodium acetate in addition to sodium chromate. By using 34 parts of soda ash (.32 mol), the reaction mass will contain .32 mol of sodium acetate and the yield of 'l-oxo cholesteryl acetate rises to about 65% of theory.

The same result may be obtained by adding 26.2 parts of anhydrous sodium acetate to the reaction mixture in Example 1.

Example 6 If, in Example 1 or 2, the sodium chromate is replaced by an equivalent amount of sodium dichromate, NazCrzOv (21.0 parts, .08 mol), the oxidation proceeds in a similar way although somewhat more rapidly, but the yield of 'l-oxo cholesteryl acetate is somewhat decreased to 50%-52% of theory.

Example 7 A solution of calcium chromate (CaCrOi) is prepared by adding 13.3 parts of chromic acid anhydride (.13 mol) to a solution of 35.2 parts of calcium acetate monohydrate (.20 mol) in parts of acetic acid anhydride and 60 parts of glacial acetic acid. The color of this solution is yellow. To the solution are added at 30 C. a solution of 42.8 parts of cholesteryl acetate (.10 mol) in 40 parts of acetic acid anhydride and 40 parts of benzene. After 24 hours at 30 C., the calcium chromate, which was originally present in theoretical quantity, has been consumed to the extent of Yield of 'l-oxo cholesteryl acetate: 55% of theory.

Example 8 By replacing in Examples 1 or 2, 42.8 parts of cholesteryl acetate with 49.0 parts of cholesteryl benzoate (.10 mol) (melting point 149-150 C., clearing point 180 C.) and conducting the oxidation at 70 C. for 60 hours, a yield of 29.3 parts of 'l-oxo cholesteryl benzoate is obtained melting point 161 C., clearing point 178 C. This is 58% of theory.

Example 9 By replacing in Examples 1 or 2, 42.8 parts of cholesteryl acetate with 40.4 parts of cholesteryl chloride (.10 mol), melt'ng point 94.5 95.0 C., and carrying out the oxidation at 40 C., 16 parts of purified l-oxo cholesteryl chloride are obtained, melting point 141-142 C. The yield is 48% of theory, based on consumed cholesteryl chloride.

Example 10 240 parts of glacial acetic acid, 11 parts of water and 24.3 parts of sodium chromate, Na2CrO4 (.15 mol, 112% of theoretical amount for oxidation) and 42.8 parts (.10 mol) of cholesteryl acetate are dissolved together in a stoppered vessel. At room temperature there occurs hardly any reaction, but by heating to 70 C. for from 60 to 80 hours the chromate is substantially consumed. The oxidation mass is worked up as described in Examples :ifior 2. the yield of Form Fcholesterylwacetate :28% of theory and 37% when making'aallowance forme'covered cholesteryl acetate. *With a .larger iexcess offso- -dium rchroma'te, sabout =37:% of 'l oxo cholesteryl acetate is directly obtained.

'Emtmt'ifile'fl 42.8 parts (.10 mol) .of cholesteryl acetateare dissolved in an oxidizing-mixture consisting of 400 parts --of glacial acetic acid, 200 parts of acetic acid anhydride, parts of sodium acetate and 21.6 parts of .sodium chromate'=(;1=33 mol or 100% of the amount calculated for the oxidation). A gentle stream of air is ipassed through the solution, which is kept'ra-t' BO C. for 48 hours, preferably 'under agitation. Titration for hexavalent chromium indicates that only 20% of the sodiumvchromatehas been :consumed. After working-up :as indicated Ein-Exl amples 1 or 2,the yield of 7- oxo 'ch'olesteryl acetate is 28.7 parts, or 155% of-itheory,'-equal*to 75% of theory after giving creditflfor recovered unused cholesteryl acetate.

' Example 12 42.8 parts (.10 mol) of cholesteryl acetate'are added to a solution of 200 partsotglacial acetic acid, 70 partsofacetic anhydride; '70parts of benzene, 6 parts of sodium acetateand 5.4 parts of theory. After makingallowance :forrecbvered unused cholesteryl acetate, they-ield is 58% of theory.

Example 13 A commercial sample' of .phyto'sterol' (rich in alpha-sito-sterol) is converted to phytosteryl acetate, melting point ll7-120 C., by boiling with acetic acid anhydrideJIfthisiproduct is oxidized under conditions analogous to Examples 1 or 2,. a crude oxidation .product -is'obtained in. very good yield, from which is isolated by means of repeated crystallizations .fromlow boiling petroleum fractions a 7-oxo phytosteryl acetate, melting point 164 166 0 which appearsto be a mixture of '7-oxo sitosteryl-acetates.

This process is particularly applicable'to the oxidation of 3-hydroxy-A -sterols to produce the '7-oxo-derivatives where the hydroxyl groupin the 3-position--hasbeenprotected by an ester group including chlorine, which-groups can be readily converted back to the hydroxyl radical. In addition to the esters-employed in the specific examples above, therprocess is: applicable to -the oxidation of any of the aliphatic esters inwhich the 3-hydroxyl group has been-esterified with any aliphatic acid containing up to 18 carbon atoms, suchas propiomc, butyricystearicyetc; also of aromatic esters otherthan the benzoate. Di-basic acids such as molanic or aromatic dibasic --acidssuch-as -phthalio may also be used. Likewise, S-hydroxy "stero-hethers such as the methyl ether may be used.

Any of the metal chromates which "are sufficient-ly soluble to be reducedin the oxidation mass may be employed. suchasthc alkali metal chromates, the ammonium chromate, calcium mate should be employed, or a 'chromate where the metal base is in excess of the chromi'c acid, whi'chfir'nay bein the form of thesalt ofan o'rga'nic acid, which 'issoluble in the reaction mass, such as *sodium acetate potassium acetate, so- -diumpropionate', sodium butyrate, eta, th'metal Fbeing any of those forming the ch'r'omates discussedabove. In any given oxidizing system the metals of the chromate and acetate or other salt =m'ay be the same or different. Preferably the acetates or'othersalt are used in the propor tion'of 0. 113010 molsper mole of cholesteryl ester. A marked increase in the yield "of the desired oxo derivative of sterol is obtaiiied in this "piec ess over the yields obtained where free chromic acidis employed, even where thechromic acid is inexcess of the n'ietal base, such as in the dich'ro'mates, or with even a lower artio of metal 'base to chromic acid. This may be due to the reduction in the acidic character of chromic acid by the metal' base which in turn modifies the oxidizing power of hexa'valentchromium. The metal "chromates may be added to the reaction all atonce, as distinguished from the pr'evious use of the chromic'acid anhydride which requires careful addition in small "increments due to "their high activity. The amountof the metal chromate required will depend on the manner in Which'the reaction is carried out, that is, whether oxygen ('or air) is'ad'oled. The amount ofchroniate used mayyary from as much as several hundred per cent in excess of'that theoretically required, to as little-as from 2% to 3% of theory when oxygen (or airl is "added to 'the"1'eaction. GrdinariIy, from 10% of theory to 200% in ex cess of theory will bepreferred.

'Eithenair or oxygen can'be' used to replace the-'chroinateto a large degree; as illustrated in the above examples. The airor oxygen "canbe incorporated by agitating the sandman-contact with air, by bubbling it through the mass orby carrying 'the reaction' out under oXygenpreSSure. This catalytic air'or oxygen 'oxidation 'requires therpresence of only small amountsof hexavalent chromium salts so that the substitiition of'part of the chromate with air or oxygen is made possible by thisinvention. 'When using chromic acid anhydride in acetic acid anhydride, the-addition of air or oxygen has no apparentefiect upon the oxidation of sterols.

Inorder to obtainthe -reaction 'mass as a homogeneous solution in which all ingredients are dissolved, it is sometimes advantageousto add an organic "solvent which is inert to chromate oxidation, such as benzene, chloro'benzene, chloroform, trichlorethylena: etc.

The best yields are obtained when thereaction is carried :out in anhydrous medium, that is, where there is an excess of acetic a'nhydride at the end of the oxidation and after liberation of'water of reaction. The beneficiareffects' o'f chromates however, ascomparedto theuse oi chromic acid itself; areevident also in a-glacial acetic acid medium and in aqueous acetic acid, ofas'low as from to acetic acid content, and much increased yields of "7-ox'o derivatives are obtained. Other organic acids, such "as propionic, butyric, etc; andtheir anhydrides,

maybe substituted .for the acetic acid 'in'this' process. v

a As illustrated in the above examples; the sterol ester can be prepared in thea'c'etic acid anhy 'atoaeet ;dride medium by refluxing for about one hour,

after which the chromate may be added and the oxidation carried out without isolation of the sterol acetate. The metal chromate may also .be produced in situ by reacting chromic acid anhydride with the metal acetate. This, however, should be carried out prior to the addition of the sterol to the reaction mass to avoid undesirable side reactions as experienced in the heretofore known processes wherein chromic acid anhydride is employed in place of the metal chromate.

The reaction may be carried out at any desired temperature within reasonable limits, the preferred temperatures being from ordinary room temperatures to 100 C. The speed of the reaction will of course depend upon various factors, as will be obvious to those skilled in the art, and the time required for completing the oxidation may vary from several hours to several days.

The present invention provides a process for producing the 7-oxo cholesteryl acetate and related sterol compounds from expensive raw materials such as cholesterol, in a yield approximately three times as great as that obtained by the previously disclosed methods wherein chromic acid anhydride is employed in slightly aqueous acetic acid, and in yields approximately twice as high as obtained where chromic acid anhydride is employed in acetic acid anhydride. It also permits more efiicient utilization of the chromic acid, for, as the eiilciency of utilization of the chromic acid in the previously described process ranges only from about to 25% of theory, in the process of the present invention the efficiency of the utilization of the chromic acid is from 40% to 60% of theory in the absence of added air or oxygen, while with the addition of air or oxygen the efficiency of utilization of the chromic acid is as high as 200% of theory, based on the chromate employed, and over 300% based on the chromate consumed.

The process of the present invention provides a safe method for carrying out the oxidation of the sterol derivatives, for, whereas the previously known methods wherein chromic acid anhydride is employed are dangerous especially because a solution of CrOs in acetic acid anhydride ignites spontaneously when warmed to 90 C. and an aqueous acetic solution decomposes violently at 120 C., the metal chromates employed in the present process in acetic acid anhydride do not ignite at any temperature. Furthermore, the oxidation of the sterol derivatives by the present method is slow, and therefore very easily controlled.

Due to the high yield of the l-oxo compounds, the lay-products are very readily removed, and in plant operation technically pure 7-oxo cholesteryl acetate, with a melting point of l56-l59 C., may be obtained directly by filtration of the oxidation mass which is of suitable purity for chemical processing without recrystallization. The methods using chromic acid anhydride have heretofore required recrystallization of the crude 7-oxo cholesteryl acetate.

I claim:

1. A process for oxidizing an ester of a 3-hydroxy-n sterol to the 7-ox0 derivative which comprises subjecting the sterol ester to oxidation in the presence of a metal chromate in a medium of the class consisting of acetic acid of at least 80% concentration, acetic anhydride and mixtures of the same, and with the addition of oxygen to the reaction mass.

2. A process for oxidizing an ester of a 3-hydroxy-A -sterol to the 7-oxo derivatives which comprises subjecting the sterol ester to oxidation with a metal chromate in a medium of the class consisting of acetic acid of at least concentration, acetic anhydride and mixtures of the same, and in the presence of added metal acetate.

3. A process for oxidizing an ester of a 3-hydroxy-A -stero1 to the 7-oxo derivative which comprises subjecting the sterol ester to oxidation in the presence of a metal chromate in a medium of the class consisting of acetic acid of at least 80% concentration, acetic anhydride, and mixtures of the same in the presence of a metal acetate and with the addition of oxygen to the reaction mass.

4. A process for oxidizing an ester of cholesterol to the 7-oxo derivative which comprises subjecting the cholesterol ester to oxidation in the presence of a metal chromate in a medium of the class consistin of acetic acid of at least 80% concentration, acetic anhydride and mixtures of the same, and with the addition of oxygen to the reaction mass.

5. A process for oxidizing an ester of cholesterol to the 7-oxo derivative which comprises subjecting the cholesterol ester to oxidation with a metal chromate in a medium of the class consisting of acetic acid of at least 80% concentration, acetic anhydride and mixtures of the same, and in the presence of added metal acetate.

6. A process for oxidizing an ester of cholesterol to the 7-oxo derivative which comprises subjecting the cholesterol ester to oxidation in the presence of a metal chromate in a. medium of the class consisting of acetic acid of at least 80% concentration, acetic anhydride and mixtures of the same in the presence of a metal acetate and with the addition of oxygen to the reaction mass.

'7. A process for oxidizing an ester of cholesterol to the '7-oxo derivative which comprises subjecting the cholesterol ester to oxidation with sodium chromate in a medium of the class consisting of acetic acid of at least 80% concentration, acetic anhydride and mixtures of the same, and with the addition of oxygen to the reaction mass.

8. A process for oxidizing an ester of cholesterol to the 'Y-oxo derivative which comprises subjecting the cholesterol ester to oxidation with sodium chromate in a medium of the class consisting of acetic acid of at least 80% concentration, acetic anhydride and mixtures of the same in the presence of added sodium acetate.

9. A process for oxidizing an ester of cholesterol to the '7-oxo derivative which comprises subjecting the cholesterol ester to oxidation with sodium chromate in a medium of the class consisting of acetic acid of at least 80% concentration, acetic anhydride and mixtures of the same in the presence of sodium acetate, and with the addition of oxygen to the reaction mass.

10. A process for oxidizing cholesteryl acetate to the 7-oxo derivative which comprises subjecting the cholesteryl acetate to oxidation with sodium chromate in acetic acid containing sufficient acetic acid anhydride so that an excess of the acetic acid anhydride is present throughout the oxidation.

11. A process for oxidizing cholesteryl acetate to the '7-oxo derivative which comprises subjecting the cholesteryl acetate to oxidation with sodium chromate in acetic acid containing suificient acetic acid anhydride so that an excess of the acetic acid anhydride is present throughout the oxidation, and passing oxygen through the reaction mass as the oxidation proceeds.

12. A process for oxidizing cholesteryl acetate to the 7-0Xo derivative which comprises subjecting the cholesteryl acetate to oxidation with sodium chromate in acetic acid containing sufiicient acetic acid anhydride so that an excess of the acetic acid anhydride is present throughout the oxidation, with an excess of sodium acetate being present in the reaction mass.

13. A process for oxidizing cholesteryl acetate to the 7-oxo derivative which comprises subjecting the cholesteryl acetate to oxidation with sodium chromate in acetic acid containing sufficient acetic acid anhydride so that an excess of the acetic acid anhydride is present throughout the oxidation, with an excess of sodium acetate being present in the reaction mass, and passing oxygen through the reaction mass as the oxidation proceeds.

14. A process for oxidizing an ester of a 3-hydroXy-A -stero1 to the 7-oxo derivative which comprises subjecting the sterol ester to oxidation with a metal chromate in glacial acetic acid and in the presence of suificient acetic anhydride to completely react with all of the water liberated in the reaction so that the reaction is carried out entirely under anhydrous conditions.

15. A process for oxidizing an ester of cholesterol to the 7-oxo derivative which comprises subjecting the cholesterol ester to oxidation with a metal chromate in glacial acetic acid and in the presence of sufficient acetic anhydride to completely react with all of the water liberated in the reaction so that the reaction is carried out entirely under anhydrous conditions.

16. A process for oxidizing an ester of cholesterol to the 7 -OXO derivative which comprises subjecting the cholesterol ester to oxidation with sodium chromate in glacial acetic acid and in the presence of sufiicient acetic anhydride to completely react with all of the water liberated in the reaction so that the reaction is carried out entirely under anhydrous conditions.

WALTER C. MEULY.

REFERENCES CITED The following references are of record in the file of this patent:

UNITED STATES PATENTS Number Nan-1e Date 2,098,984 Windaus Nov. 16, 1937 2,323,584 Schoeller July 6, 1943 FOREIGN PATENTS Number Country Date 467,376 Great Britain June 16, 1937 

1. A PROCESS FOR OXIDIZING AN ESTER OF A 3-HYDROXY-$5-STEROL TO THE 7-OXO DERIVATIVE WHICH COMPRISES SUBJECTING THE STEROL ESTER TO OXIDATION IN THE PRESENCFE OF A METAL CHROMATE IN A MEDIUM OF THE CLASS CONSISTING OF ACETIC ACID OF AT LEAST 80% CONCENTRATION, ACETIC ANHYDRIDE AND MIXTURES OF THE SAME, AND WITH THE ADDITION OF OXYGEN TO THE REACTION MASS. 